Continual regeneration of the intestinal lining relies on stem and progenitor cells in sub-mucosal crypts, and intestinal disorders, including cancer, reflect dysfunction of these cells. Lgr5-expressing intestinal stem cells (ISC) replicate daily, producing bipotential progenitors (BP) that choose between alternative cell fates: enterocyte or secretory. This key cell fate decision is incompletely understood and the intestinal epithelium is a powerful model to investigate intermediate cell states. Therapeutic modulation of signaling pathways in the digestive tract also requires deeper appreciation of the underlying principles and mechanisms. Recent evidence reveals that crypt cells are considerably plastic, and in work supported by parent R01DK081113, we showed that broadly permissive chromatin underlies this plasticity. Aim 1 of the parent R01 seeks to determine tissue-restricted enhancer elements and binding sites for the transcription factor ATOH1 in secretory progenitors because ATOH1 allocates cell fate by lateral inhibition. As mixed cell populations can and have addressed the question, this Aim does not need single-cell resolution. The second Aim, however, investigates early ISC-derived BP, which are difficult to identify or characterize because they lack known molecular markers and information is lost in mixed-cell populations. Showing that single-cell analysis is well suited to advance project goals and address fundamental questions of broad interest, our preliminary single-cell gene expression analyses indicate that putative BP are a distinct subpopulation of Lgr5hi cells. Therefore, the first Specific Aim in this proposal is to identify and characterize these and other functionally distinct Lgr5+ ISC subpopulations. We will use single-cell RT-PCR to determine accurate transcript levels of ~275 genes carefully chosen to report cell lineage, replication status, surface markers, and differentiation states. We will alo measure these genes in specified secretory and enterocyte progenitors, purified by methods developed under the parent R01. We will build new analytical tools to determine cell relationships and hierarchies; preliminary data show that our experimental and computational approaches are feasible. Beyond gene expression, cells differ in surface markers and pathways for rapid intracellular signaling, and our second Specific Aim is to identify the basis for crypt cll heterogeneity in different signaling pathways using time-of-flight mass cytometry (CyTOF). Preliminary data reveal that AKT/S6 kinase signaling is confined to Lgr5hi ISC, quickly diminishing in BP and specified progenitors. We will substantively extend single-cell analysis by CyTOF to interrogate other signaling pathways and surface markers. For both Aims, we will rigorously test new hypotheses about cell subpopulations using crypt organoid cultures, and we propose new computational methods for integrated analysis of single-cell data from our two orthogonal approaches. This robust 2-tiered investigation of single cells will inform understanding of intestinal self- renewal and develop a strategy for broad application toward other tissues and biomedical problems.